The capacity to evaluate the teratoma forming potential of differentiated embryonic stem cell populations represents an urgent need before widespread therapeutic application of these cells is possible. Researchers at Case Western Reserve University and BioInVision, Inc. will join together to create platform technologies for validating existing embryonic stem cell separation and purification paradigms (such as antibody labeling and Fluorescent Activated Cell Sorting), as well as future purification paradigms. The biologic platform consists of embryonic stem cells which report one fluorescent marker (red) when undifferentiated, and another marker (green) when differentiated down the endothelial lineage. Sorting will be evaluated in vitro by plating green cells, growing those cells in long-term culture, and determining any appearance of red undifferentiated cells. To assess cell in vivo, we will use BioInVision's unique cryo-imaging technology which can detect single fluorescent cells in a mouse. Briefly, the cryo-imaging system consists of a large stage, motorized cryostat with special features for imaging;a modified, bright-field/fluorescence microscope;and a robotic xyz imaging system positioner, all of which are fully automated by a control system to allow sectioning and tiled imaging of an entire mouse. The basic experimental paradigm will consist of sorting differentiated (green) cells and implanting them via tail injection. Days later, cryo-imaging will be used to identify teratomas and/or undifferentiated (red) cells with the potential of forming a teratoma. The cryo-imaging platform represents a distinct advantage over other imaging methods, such as bioluminescence, MRI, and PET, in that it is capable of detecting not only large overt teratomas, but also finding single cells which may have the capacity to form teratomas in the future. PUBLIC HEALTH RELEVANCE: This proposal is critically concerned with the issue of public health in that its long-term goal is to provide a means for evaluating the effectiveness of embryonic stem cell selection and purification strategies prior to such cells being used therapeutically. The capacity for teratoma formation is the main barrier preventing use of embryonic stem cells clinically, for diseases such as diabetes and Parkinson's Disease and spinal cord injury.